Determination of triglycerides

ABSTRACT

Triglycerides are determined by enzymatic saponification with lipase and measurement of the liberated glycerol by carrying out the saponification in the presence of carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate; reagents are provided for carrying out the process and comprise a buffer and, as a saponification agent, lipase, carboxylesterase and the alkali metal or alkaline earth metal alkyl sulfate, and a glycerol detection system.

United States Patent .19.

Wahlefeld et al.

[ Jan. 21, 1975 DETERMINATION OF TRIGLYCERIDES [75] Inventors: August Wilhelm Wahlefeld,

Weilheim, Obb; Hans Mollering, Tutzing, Obb; Wolfgang Gruber, Garatshausen NR.; Erich Bernt, Munich; Peter Roeschlau, Tutzing, Obb, all of Germany [73] Assignee: Boehinger Mannheim GMBH,

Mannheim, Germany [22] Filed: May 30, 1973 [21] Appl. No.: 365,355

[30] Foreign Application Priority Data June 19, 1972 Germany 2229849 [52] US. Cl. l95/l03.5 R, 195/63 [51] Int. Cl C07g 7/02, C12k l/OO [58] Field of Search 195/62, 66, 63, 68, 103.5 R; 73/230 B [56] References Cited UNITED STATES PATENTS 3,703,591 11/1972 Bucolo et al 195/1035 R 3,759,793 9/1973 Stork et al. l95/1()3.5 R

Primary ExaminerDavid M. Naff Attorney, Agent, or Firm-Burgess, Dinklage &

Sprung [57] ABSTRACT 17 Claims, No Drawings DETERMINATION OF TRIGLYCERIDES The present invention is concerned with a process and reagent for the determination of triglycerides by the saponification of glycerides and determination of the glycerol thereby liberated.

The determination of triglycerides plays an increasingly important part in foodstuff analysis and also in medical diagnosis, especially in the diagnosis of hyperlipemias in clinical chemistry.

According to one known determination process, the triglycerides are first saponified with an alcoholic solution of an alkali metal hydroxide and the glycerol formed is then determined, the determination preferably being carried out by enzymatic methods. In this case, the glycerol is phosphorylated with adenosine triphospate (ATP) in the presence of glycerokinase (GK) to give glycerol-l-phosphate and adenosine diphosphate (ADP). The ADP formed is, in turn, converted by phosphoenol pyruvate (PEP), in the presence of pyruvate kinase, into pyruvate and ATP. The pyruvate hereby formed is hydrogenated by nicotinamide-adenine-dinucleotide in the reduced form (NADH), in the presence of lactate dehydrogenase (LDH), to give lactate, the NADH thereby being oxidized to nicotinamide-adeninedinucleotide (NAD). The amount of NADH utilized by the reaction is equivalent to the amount of glycerol. NADH can easily be determined quantitatively on the basis of its absorption at 366, 340 or 344 nm.

An important disadvantage of this known process is the saponification with an ethanolic solution of an alkali metal hydroxide. This saponification step makes the otherwise specific, precise and readily carried out method laborious and time-consuming since the saponification alone requires 20 to 30 minutes at a temperature of about 70C. Subsequently, neutralization is necessary, as well as centrifuging, before the actual glycerol determination can be commenced.

This disadvantage can be overcome in the manner described in our British Patent Specification No. 1,295,992 in which the triglycerides are saponified enzymatically, using a lipase from Rhizopus arrhizus. A surprising feature of this method was that a lipase had been found which can split the triglycerides completely in an aqueous buffer solution within an acceptable period of time to give fatty acids and glycerol. Other lipases, especially the known pancreas lipase, proved to be unsuitable.

A disadvantage of the enzymatic splitting is, however, that the saponification still takes quite a long time and, in addition, necessitates the use of considerable amounts of the very expensive enzyme. In order to achieve useful reaction times, it is necessary to use about 1 mg. of the enzyme per test. Furthermore, the reaction time is more than 30 minutes and thus is scarcely suitable for routine laboratory investigations, especially in the case of tests which have to be carried out frequently. In addition, the liberated fatty acids form insoluble soaps with calcium and magnesium ions which, in turn, give rise to turbidity and thus a falsification of the measurement results if centrifuging is not carried out.

It is, therefore, an object of the present invention to overcome these disadvantages and'to provide a process for the determination of triglycerides by means of an enzymatic saponification in which thenecessary amount of lipase, as well as the necessary expenditure of time, are considerably reduced and, in addition, the necessity of separating precipitated soaps is overcome.

Thus, the present invention provides a method for the determination of triglycerides by enzymatic saponification by means of a lipase and measurement of the liberated glycerol, which process comprises carrying out the saponification in the presence of carboxylesterase and of an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains 10 to 15 carbon atoms.

The new process according to the present invention is preferably carried out in the presence of serum albu- Surprisingly, the reagent combination used according to the present invention enables the amount of lipase necessary for the saponification to be very considerably reduced from about 1 mg. per test 20 pg. per test, with a simultaneous reduction of the reaction temperature to ambient temperature and of the period of the reaction.

It is preferred to use a lipase from Rhizopus arrhizus. As carboxylesterase, EC 3.1.1.1 carboxyl ester hydrolase, there is preferably used a mammalian liver preparation, especially a pig liver esterase. However, other carboxylesterases can also be used. An amount of about pg. esterase per test has proved to be completely sufficient.

Amongst the alkyl sulfates, the alkali metal salts are preferred because they do not form insoluble soaps with the liberated fatty acids. In the case of the preferred embodiment of the process according to the present invention, using serum albumen, the alkaline earth metal alkyl sulfates can also be used. The dodecyl sulfates are preferred because they accelerate the saponification of the triglycerides under these conditions the most (factor 5). The alkyl sulfate is effective in an amount of as small as 0.01 mg./ml in the test batch. Amounts above 1.0 mg./ml. can lead to a disturbing foam formation and slight inhibition and are, therefore, preferably not used.

The process according to the present invention is preferably carried out at a pH between 6 and 9 and more preferably at at pH of 7 to 8.5. This has the advantage that, in the case of detection of the glycerol formed by the known reaction using ATP, GK, PEP, PK, NADH and LDH, no rebuffering is essential so that the saponification and measurement of the glycerol formed can be carried out in a single reaction batch.

In the case of the preferred embodiment of the process according to the present invention, with the addition of serum albumen, preferably of bovine serum albumen, a turbidity due to precipitated soaps is avoided so that such soaps do not have to be separated off, for example, by centrifuging. The serum albumen is preferably used in a concentration of between 0.1 and 2.0 mg./ml. test batch.

Cobalt and/or magnesium ions possess a certain activating effect on the lipase and esterase and can, there fore, also be added for further acceleration of the reaction.

Any buffer can be used which is effective in the abovegiven pH range, with the exception of phosphate buffers. Examples of suitable buffers include triethanol-amine buffer, tris buffer, imidazole buffer, veronal buffer and glycine buffer, as well as, but less preferably, amediol buffer, borate buffer and collidine buffer.

The new reagent according to the present invention for carrying out the process of the present invention comprises a system for the detection of glycerol and, in addition, lipase, carboxylesterase, an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains to carbon atoms, and optionally also serum albumen.

In principle, any known system can be used for the detection of glycerol in the reagent according to the present invention. A preferred detection system comprises NADH, ATP, PEP, LDH, PK and GK, as well as magnesium ions and buffer. This detection system can be readily used in the presence of the saponification agent combination according to the present invention and has, in addition, the advantage that the components do not disturb each other and all the enzymes are active in the same pH range.

Within the scope of the preferred reagent combination, an especially preferred reagent consists of:

0.1 to 10.0 mg/ml. lipase from Rhizopus arrhizus 0.5 to 20.0 mg. carboxylesterase 0.01 to 0.2 mg. alkyl sulfate (preferably sodium dodecyl sulfate) 1 to mM NADH 10 to 100 mM ATP 2 to 20 mM PEP 0.5 to 5 mg. LDH

0.2 to 5 mg. PK

0.05 to 10 mg. GK

0.1 to 2.0 mg. serum albumen 3 to 30 mM magnesium ions optionally 0.5 to 1.0 mM cobalt ions and 0.03 to 0.3M buffer of pH 6 to 9.

The above-given amounts each refer to 1 ml. of buffer solution of the given concentration. As buffer, the reagent combination preferably contains a 0.1M triethanolamine buffer of pH 7 to 8.5

As already mentioned, the process and reagent according to the present invention permit an extraordinary acceleration and simplification of the triglyceride determination. In comparison with the known process with saponification using ethanolic potassium hydroxide solution, the time of the determination is reduced from more than an hour to less than 30 minutes. At the same time, numerous pipetting steps, heating of the saponification batch, neutralizing and centrifuging off of precipitate and rendered superfluous.

In comparison with the previously known process using enzymatic saponification, a considerable acceleration is achieved, with a simultaneous very considerable reduction of the amount of enzyme necessary.

The following Examples are given for the purpose of illustrating the present invention:

EXAMPLE 1 There is prepared a storage-stable reagent, consisting of 5 components which are mixed prior to use, and is then storage-stable in a mixed state for l to 2 days:

Component 1: 0.1M triethanolamine buffer, pH 7.6, containing 3 mM magnesium sulfate, 1.5 mg. bovine serum albumen/ml. and 0.1 mg. sodium dodecyl sulfate/ml.

Component 2: solution of6 mM NADl-l, 33 mM ATP and 11 mM PEP in distilled water Component 3: crystalline suspension of 2 mg. LDH/ml. and 1 mg. PK/ml. (commercially available) Component 4: solution of 0.2 mg. lipase from Rhizopus arrhizus/ml. and 4.0 mg. carboxylesterase/ml.

Component 5: crystalline suspension of 2 mg. GK/ml. 2.9 ml. Component 1,0.1 ml. Component 2 and 0.02 ml.

Component 3 were mixed and warmed to 25C. 0.1 ml. serum, which contains the triglycerides to be determined, was then admixed therewith and incubated for 5 minutes at the given temperature. 0.1 ml. Component 4 was then admixed, the mixture was maintained for 15 to 20 minutes at the given temperature and subsequently the extinction was read off in a photometer at 366 nm or at 340 nmv The valve read off is E Subsequently, 0.02 ml. Component 5 was added to the test sample and, after 10 minutes, the extinction was again read off. The measurement value obtained was After a further 10 minutes, a further reading was made which was E The results were evaluated as follows:

A r 2) 2 a) EXAMPLE 2 The process described in Example 1 was repeated with the use of a triglyceride standard solution. In experiment 1, the carboxylesterase and dodecyl sulfate was omitted, in experiment 2 the dodecyl sulfate was omitted, in experiment 3 the carboxylesterase was omitted and in experiment 4 the complete reagent was used. The results obtained, expressed as percentages of the triglycerides found during the above-given period of reaction, are set out in the following Table:

experiment esterase dodecyl sulfate triglycerides found The above results that, under the conditions used with regard to lipase concentration, only the reagent combination according to the present invention is suitable for a quantitative determination.

lt will be understood that the specification and examples are illustrative but not limitatve of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. Reagent for the determination of triglycerides by enzymatic saponification which reagent comprises a saponification agent, a buffer, and a system for the de tection of glycerol, wherein the saponification agent comprises a lipase obtained from Rhizopus arrhizus, carboxylesterase, and an alkali metal or alkaline earth metal alkyl sulfate, wherein the alkyl group contains from 10 to 15 carbon atoms.

2. Reagent as claimed in claim 1 wherein the said carboxylesterase is a pig liver esterase.

3. Reagent as claimed in claim 1 wherein the said system for the detection of glycerol comprises nicotinamideadenine-dinucleotide in reduced form, adenosine triphosphate, phosphoenol pyruvate, lactate dehydrogenase, pyruvate kinase, glycerokinase, cobalt and/or magnesium ions and a buffer.

4. Reagent as claimed in claim 1 wherein the buffer is a 0.1M triethanolamine buffer of pH 7 to 8.5.

5. Reagent as claimed in claim 1 wherein the alkyl sulfate is an alkali metal sulfate.

6. Reagent as claimed in claim 1 wherein the alkyl sulfate is an alkaline earth metal alkyl sulfate.

7. Reagent as claimed in claim 1 wherein the reagent additionally contains serum albumin.

8. Reagent as claimed in claim 1 additionally comprising 0.5 to 1.0 mM of cobalt ions.

9. Reagent as claimed in claim 1 comprising:

0.1 to 10.0 mg./ml. lipase from Rhizopus arrhizus 0.5 to 20.0 mg./ml. carboxylesterase 0.01 to 0.2 mg./ml. sodium dodecyl sulfate 1 to 20 mM nicotinamide-adenine-dinucleotide in reduced form to 100 mM adenosine triphosphate 2 to mM phosphoenol pyruvate 0.5 to 5 mg./ml. lactate dehydrogenase 0.2 to 5 mg./ml. phosphoenol kinase 0.05 to 10 mg. glycerokinase 0.1 to 2.0 mg./ml. serum albumin 3 to 30 mM magnesium ions and 0.03 to 0.3 M buffer solution of pH 6 to 9.

10. Method for the determination of triglycerides by enzymatic saponification, which method comprises saponifying a sample containing triglycerides with a saponification agent comprising a lipase obtained from Rhizopus arrhizus, carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate, wherein the alkyl radicals are of from 10 to 15 carbon atoms, and measuring the liberated glycerol.

11. Process as claimed in claim 10 wherein the carboxylesterase is a pig liver esterase.

12. Method as claimed in claim 10 wherein the saponification is carried out in the additional presence of serum albumin.

13. Method as claimed in claim 10 wherein the saponification is carried out at a pH from 6 to 9.

14. Method as claimed in claim 10 wherein the saponification is carried out at a pH between 7 and 8.5.

15. Method as claimed in claim 10 wherein the said alkyl sulfate is used in an amount of from 0.01 to 1.0 mg./ml. of reagent basis.

16. Method as claimed in claim 12 wherein the serum albumin is used in an amount of 0.1 to 2.0 mgs./ml. of reagent solution.

17. Method as claimed in claim 10 wherein the determination is carried out with a combined reagent comprising the saponification agent, the system for the detection of glycerol, and a buffer comprising:

0.1 to 10.0 mg./ml. lipase from Rhizopus arrhizus 0.5 to 20.0 mg./ml. carboxylesterase 0.01 to 0.2 mg./ml. sodium dodecyl sulfate 1 to 20 mM nicotinamide-adenine-dinucleotide in reduced form 10 to mM adenosine triphosphate 2 to 20 mM phosphoenol pyruvate 0.5 to 5 mg./ml. lactate dehydrogenase 0.2 to 5 mg./ml. phosphoenol kinase 0.05 to 10 mg. glycerokinase 0.1 to 2.0 mg./ml. serum albumin 3 to 30 mM magnesium ions and 0.03 to 0.3 M buffer solution of pH 6 to 9. 

1. Reagent for the determination of triglycerides by enzymatic saponification which reagent comprises a saponification agent, a buffer, and a system for the detection of glycerol, wherein the saponification agent comprises a lipase obtained from Rhizopus arrhizus, carboxylesterase, and an alkali metal or alkaline earth metal alkyl sulfate, wherein the alkyl group contains from 10 to 15 carbon atoms.
 2. Reagent as claimed in claim 1 wherein the said carboxylesterase is a pig liver esterase.
 3. Reagent as claimed in claim 1 wherein the said system for the detection of glycerol comprises nicotinamideadenine-dinucleotide in reduced form, adenosine triphosphate, phosphoenol pyruvate, lactate dehydrogenase, pyruvate kinase, glycerokinase, cobalt and/or magnesium ions and a buffer.
 4. Reagent as claimed in claim 1 wherein the buffer is a 0.1M triethanolamine buffer of pH 7 to 8.5.
 5. Reagent as claimed in claim 1 wherein the alkyl sulfate is an alkali metal sulfate.
 6. Reagent as claimed in claim 1 wherein the alkyl sulfate is an alkaline earth metal alkyl sulfate.
 7. Reagent as claimed in claim 1 wherein the reagent additionally contains serum albumin.
 8. Reagent as claimed in claim 1 additionally comprising 0.5 to 1.0 mM of cobalt ions.
 9. Reagent as claimed in claim 1 comprising: 0.1 to 10.0 mg./ml. lipase from Rhizopus arrhizus 0.5 to 20.0 mg./ml. carboxylesterase 0.01 to 0.2 mg./ml. sodium dodecyl sulfate 1 to 20 mM nicotinamide-adenine-dinucleotide in reduced form 10 to 100 mM adenosine triphosphate 2 to 20 mM phosphoenol pyruvate 0.5 To 5 mg./ml. lactate dehydrogenase 0.2 to 5 mg./ml. phosphoenol kinase 0.05 to 10 mg. glycerokinase 0.1 to 2.0 mg./ml. serum albumin 3 to 30 mM magnesium ions and 0.03 to 0.3 M buffer solution of pH 6 to
 9. 11. Process as claimed in claim 10 wherein the carboxylesterase is a pig liver esterase.
 12. Method as claimed in claim 10 wherein the saponification is carried out in the additional presence of serum albumin.
 13. Method as claimed in claim 10 wherein the saponification is carried out at a pH from 6 to
 9. 14. Method as claimed in claim 10 wherein the saponification is carried out at a pH between 7 and 8.5.
 15. Method as claimed in claim 10 wherein the said alkyl sulfate is used in an amount of from 0.01 to 1.0 mg./ml. of reagent basis.
 16. Method as claimed in claim 12 wherein the serum albumin is used in an amount of 0.1 to 2.0 mgs./ml. of reagent solution.
 17. Method as claimed in claim 10 wherein the determination is carried out with a combined reagent comprising the saponification agent, the system for the detection of glycerol, and a buffer comprising: 0.1 to 10.0 mg./ml. lipase from Rhizopus arrhizus 0.5 to 20.0 mg./ml. carboxylesterase 0.01 to 0.2 mg./ml. sodium dodecyl sulfate 1 to 20 mM nicotinamide-adenine-dinucleotide in reduced form 10 to 100 mM adenosine triphosphate 2 to 20 mM phosphoenol pyruvate 0.5 to 5 mg./ml. lactate dehydrogenase 0.2 to 5 mg./ml. phosphoenol kinase 0.05 to 10 mg. glycerokinase 0.1 to 2.0 mg./ml. serum albumin 3 to 30 mM magnesium ions and 0.03 to 0.3 M buffer solution of pH 6 to
 9. 